Implications of land management on soil

Tropical Ecology 53(2): 215-224, 2012
© International Society for Tropical Ecology
www.tropecol.com

ISSN 0564-3295

Implications of land management on soil microbial communities and
nutrient cycle dynamics in the lowland tropical forest
of northern Costa Rica
1

2

3

3

K. HAFICH , E. J. PERKINS , J. B. HAUGE , D. BARRY & W. D. EATON

4*

1

Department of Earth and Planetary Sciences, University of New Mexico,
Albuquerque, NM 87131-001, USA
2
Biology Department, University of Puget Sound, Tacoma, WA 98416-1088, USA
3
Peninsula College, Center of Excellence in Environmental Science and Natural Resources,
and Western Washington University Huxley College of the Environment at Peninsula
College, Port Angeles, WA 98362 USA
4
School of Environmental and Life Sciences, Kean University, Union, New Jersey 07083, USA

Abstract: The long-term effects of reforestation versus maintained grassland on microbial
community structure and nutrient cycling provide a valuable perspective on ecosystem health
and carbon sequestration potential of tropical soils in the heavily deforested Northern Zone of
Costa Rica. The soil from the secondary forests in this area had greater levels of phosphate,
inorganic nitrogen, organic carbon, respiratory activity, abundance and diversity of
Basidomycete rDNA, abundance of fungal rDNA, and lower abundance but greater diversity of
Rhizobium rDNA, and less abundance of nifH gene DNA than soils from adjacent grasslands of

the same age. Critical correlations were observed between the abundance of Basidiomycete
rDNA and laccase gene with the levels of phosphate, microbial biomass, organic carbon use
efficiency, and percent water saturation. These data suggest a trend towards the secondary
forest soils becoming more fungal-dominant, with greater microbial activity, greater nitrogen
mineralization activity and more efficient use of carbon. This project provides some of the first
evidence that the management strategy of regeneration of secondary forests results in more
complex soil ecosystems, with greater potential for carbon sequestration than the maintained
grasslands.
Resumen: Los efectos a largo plazo de la reforestación contra los del mantenimiento de
pastizales sobre la estructura de la comunidad microbiana y el reciclaje de los nutrientes
ofrecen una perspectiva valiosa sobre la salud del ecosistema y el potencial para secuestrar
carbono de los suelos tropicales en la zona norte de Costa Rica, fuertemente deforestada. El
suelo de los bosques secundarios en esta área tuvo niveles más altos de fosfato, nitrógeno
inorgánico, carbono orgánico, actividad respiratoria, abundancia y diversidad de ADNr de
basidiomicetos, abundancia de ADNr fúngico, y una menor abundancia pero una mayor
diversidad de ADNr de Rhizobium, así como una abundancia menor de ADN del gen nifH que
los suelos de pastizales contiguos de la misma edad. Se observaron correlaciones críticas entre
la abundancia de ADNr de basidiomicetos y del gen de la lacasa con los niveles de fosfato, la
biomasa microbiana, la eficiencia del uso del carbono orgánico y el porcentaje de saturación de
agua. Estos datos sugieren que los bosques secundarios tienden a estar más dominados por

hongos y a tener una mayor actividad microbiana, una mayor actividad de mineralización del
nitrógeno y un uso más eficiente del carbono. Este proyecto proporciona algunas de las primeras
*

Corresponding Author; e-mail: weaton@kean.edu

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LAND MANAGEMENT AND SOIL MICROBIAL COMMUNITIES

evidencias de que la estrategia de manejo de la regeneración de los bosques secundarios resulta
en ecosistemas edáficos más complejos, con un mayor potencial para el secuestro de carbono,
que el mantenimiento de los pastizales.
Resumo: Os efeitos de longo prazo da reflorestação, versus manutenção da pastagem na
estrutura da comunidade microbiana e no ciclo de nutrientes, proporcionam uma perspectiva
valiosa sobre a sanidade do ecossistema e no potencial de sequestração de carbono de solos
tropicais na região fortemente desflorestada na zona norte da Costa Rica. O solo das florestas
secundarias nesta área apresentava altos teores de fosfato, azoto inorgânico, carbono orgânico,
atividade respiratória, abundância e diversidade de rDNA de Basidiomicetas, abundância de
rDNA fúngico e pouca abundância mas grande diversidade de rDNA de Rhizobium e menos

abundância do gene nifH do DNA do que nos solos das pastagens adjacentes da mesma idade.
Foram observadas correlações críticas entre a abundância do rDNA de Basidiomiceta e do gene
da lacase com os níveis de fosfato, da biomassa microbiana, a eficiência de uso do carbono
orgânico, e a percentagem da saturação de água. Estes dados sugerem a tendência para que os
solos da floresta secundária se apresentarem com uma maior dominância da componente
fúngica, com maior atividade microbiana, maior atividade de mineralização do azoto e um uso
mais eficiente do carbono. Este projeto providenciou alguma da primeira evidência de que a
estratégia de gestão da regeneração resulta em ecossistemas de solo mais complexos, com maior
potencial para a sequestração de carbono do que as pastagens mantidas.

Key words: Carbon sequestration, Costa Rica, Maquenque National Wildlife Refuge,
nitrogen, nutrient cycling, soil microbial communities, tropical reforestation.

Introduction
The effect of tropical rainforest deforestation
has reached across the globe, at scales ranging
from atmospheric to microbial communities and
geochemical properties of tropical soils. Globally,
deforestation has been documented for several
decades as contributing to an atmospheric rise in

greenhouse gas levels (e.g. Fearnside 2000), changes in release of trace gases (e.g. Keller et al. 1993)
and in hydrological processes (e.g. Meher-Homji
1991), and significant losses of biodiversity at
many trophic levels (e.g. Ehrlich & Wilson 1991).
After forests are cleared, whether land is managed grassland, allowed to regenerate as secondary
forest, or used for other purposes, has substantial
impact on vegetation, nutrient availability, and
microbial communities (Cleveland et al. 2003;
Decaëns et al. 2006; Nüsslein & Tiedje 1999; Reiners
et al. 1994).
Costa Rica experienced significant deforestation in the twentieth century, as well as
reforestation in the last 40 - 50 years (Chassot et
al. 2001, 2005; Monge et al. 2002, 2003). Between
1941 - 1983, Sader & Joyce (1988) estimate that 83 %
of forested land in Costa Rica was cleared, with

disproportionally more habitat loss in the tropical
lowlands. From the mid-1980s to the mid 1990s,
reforestation in the Northern lowlands of Costa
Rica balanced out deforestation, with 7.3 % of the

landscape being reforested and 7.9 % of land being
converted from forest to pastures (Powers 2004).
Wardle (2006) recognized the need to identify
the linkages between above and below-ground
diversity, the roles of nutrient resource availability, and terrestrial habitat properties, yet few
studies have connected these together in habitat
assessments, especially within tropical ecosystems.
Chazdon et al. (2007) discussed the rates of changes in vegetation occurring in some of the lowland
forests of Costa Rica following the original disturbance leading to the development of older, wellfunctioning secondary forests. However, the composition and function of the microbial community
in such secondary forests in this part of Costa Rica
has not been addressed to date. Many studies have
shown that the assessment of microbial community and nutrient cycle dynamics can be used as
indicators of a healthy soil ecosystem with potential value for assessment of ecosystem management, restoration, and conservation strategies employed in the secondary forests of the tropics (e.g.

HAFICH et al.

Buckley & Schmidt 2003; Hartmann & Widmer
2006; Ibekwe et al. 2007; Wardle et al. 2004). As
reforestation becomes more common in the tropics,
the examination of the microbial community and

associated nutrient cycle components could be used
to monitor soil ecosystem development to assess
the efficacy of this and other land management
practices. Thus, the objective of this study was to
compare the long-term effects of forest clearing
followed by grassland maintenance versus secondary forest regeneration on components of the soil
ecosystems in the Northern Zone of Costa Rica.

Materials and methods
Study sites and sample collection
The study sites used were within the Maquenque National Wildlife Refuge (MNWLR) in Costa
Rica, which is located in Northeast region of Costa
Rica, about 15 km south of the Nicaraguan border
(10° 27'05.7"N, 84° 16' 24.32"W ). The sites had
been primary forest that was cleared in the early
1980’s. Part of the land was allowed to regenerate
into a mixed species secondary forest and part was
established as pasture for cattle. The cattle were
removed from the pasture beginning in 1991, and
the pasture was then maintained as grassland.

Four plots of 1000 m2 were established in the
secondary forest and the grasslands, and divided
into ten 10 x 10 m subplots. Within each subplot,
eight randomly located 2 cm diameter x 15 cm
deep cores were collected and then composited by
subplot on two consecutive days in July, 2009. This
resulted in four discrete soil samples per habitat
type, made up of 80 soil cores (10 subplots x 8
cores) that were then analyzed. Soil was collected
using sterile technique to avoid cross contamination between plots. Separate samples were
collected at each subplot for bulk density determination, and the percent saturation and pH were
also determined at 10 randomly located sites
within each plot using a Kelway HB-2 Soil and pH
meter (Wyckoff, NJ, USA). Samples were mixed
and sieved at 10 mm to remove rocks, insects and
plant matter. All data were adjusted for soil moisture
levels, dry weight, and bulk density to standardize
results to a common soil volume (Cleveland et al.
2003; Powers 2004).

Nutrient analysis
Nutrient analyses were conducted within two
days of soil collection. Nitrate (as NO3-N) and
ammonium (as NH4-N) content were measured

217

using 50 mL of 2M KCl extraction of 10 g of soil
(Alef & Nannapieri 1995) followed by ammonium
salicylate and cadmium reduction spectrophotometric methods using the HACH DR 2700 system
(Hach Company, Loveland, Colorado, 80539-0389;
HACH methods 8155 and 8192 respectively). The
total mineral nitrogen (TMN) levels were estimated as the sum of the NO3-N and NH4-N values.
Phosphate (PO4) was measured after Bray 1
extraction from 2 g of soil using the molybdenate
reduction method (HACH method # 8048) and the
HACH DR 2700 system.
The microbial biomass C (Cmic) was determined
for each sample by the fumigation-extraction
method (Jenkinson 1988) as the difference between

the 0.5 M K2SO4-extracted soil dissolved organic
carbon (DOC) levels in ethanol-free chloroformfumigated and unfumigated 10 g soil subsamples.
The DOC levels were determined by dry combustion analysis at the CATIE labs in Turrialba,
Costa Rica, using the methods of Anderson &
Ingram (1993) and an autoanalyzer (Alliance
Instruments). The rate of respiration was determined as CO2 released using a Qubit SR1LP Respiration system (Kingston, ON, Canada). The
microbial metabolic quotients (qCO2, as a ratio of
CO2 from respiration/Cmic) and the ratio of Cmic/
SOC were determined to estimate the efficiency of
utilization of organic C (Anderson 2003).

DNA analysis
DNA was extracted from soil samples within
five days of collection using the Power Soil DNA
Isolation kit (MO BIO, Carlsbad, CA). Three
replicates were used for each of the four plots per
habitat type, using 0.3 g of soil for each extraction,
after which the replicates were pooled for PCR
analysis. DNA was stored at 4 °C until use. Endpoint PCR (using an Applied Biosystems, Foster
City, CA 9700 Thermal Cycler) was used to

identify the presence of target genes in each
sample. The primers referenced in Table 1 and
AmpliTaq Gold (Applied Biosystems, Foster City,
CA) complete Master Mix were used for the PCR
assays, along with the suppliers recommend PCR
temperature schedule, except that for laccase,
nifH, Basidiomycete ITS, and 16S Rhizobium PCR
assays, the annealing temperature was 55 °C; and
the 18s fungal rRNA annealing temperature was
48 °C. The relative abundance (RA) of each target
gene was estimated using a quantitative PCR
assay using the above primers and conditions and
a MJ Research Opticon One thermal cycler and

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LAND MANAGEMENT AND SOIL MICROBIAL COMMUNITIES

Table 1. PCR primers used to identify the presence
and diversity of target genes in soils from secondary
forests and adjacent grasslands in the Northern Zone
of Costa Rica.
Gene
Primer
Sequence
nifH gene nifHf TAC GGN AAR
GGS GGN ATC
GGC AA
nifHI AGC ATG TCY
TCS AGY TCN
TCC A

Reference
Grange et al.
2007

Rhizo63f
bium 16S
Rhiz12
44r

Singh et al.
2006

Fungal
18S

Basidiomycete
ITS
Laccasegene

CTC GCT GCC
CAC TGT CAC
AGG CCT AAC
ACA TGC AAG
TC

EF4

GGA AGG GRT Smit et al.
GTA TTT ATT AG 1999
EF3
TCC TCT AAA
TGA CCA ACT
TTG
ITS1F CTTGGTCATTTA Dickie &
GAGGAAGTAA FitzJohn 2007
ITS4
TCCTCCGCTTAT
TGATATGC
Lac2f CGC ATC ATC
Litvintseva &
TTT TGT GCT CC Henson 2002
Lac2r AGC GCA ACT
ACG ACG AGG A

*Boldface letters signify degenerate
A+C+G+T; R, A+G; S, G+C; Y, C+T.

positions.

N,

Table 2. Restriction
endonucleases
used
in
Restriction Fragment Length Polymorphism (RFLP)
assays to estimate the diversity of target genes in
soils from secondary forests and adjacent grasslands
in the Northern Zone of Costa Rica.
Gene

Enzymes

nifH gene

HaeIII/Mspl/Mbol/Hhal

Rhizobium 16S r RNA

Hhal/Mspl

Fungal 18S rRNA

HinfI/TaqI

Laccase gene
Basidiomycete ITS

Hhal/Mspl/TaqI
HaeIII/HinfI/AluI/Taq

Thermo Scientific SYBR Green Master Mix in
which the abundance of the PCR product DNA
from the soil samples was compared to known
concentrations of purified target gene DNA (6.5 to

28.4 ng µL-1 of cloned target gene DNA with
sequences confirmed in GenBank), from which the
target DNA concentrations in each sample were
determined, and the RA calculated. The percent of
the total RA that each target gene represented
within a sample was determined and reported. A
restriction fragment length polymorphism (RFLP)
assay was performed on the PCR amplified DNA
from each sample to provide a preliminary estimate
of the target gene diversity. The restriction endonucleases were from Fermentas, Inc. (Burlington,
ON, Canada) and are listed in Table 2. The
digestions were performed using the methods suggested by the vendor and a known concentration of
PCR product (approximately 0.2 µg). The resulting
size and quantity (per µg of starting PCR product)
of each DNA band (in 2 % agarose electrophoresis)
were determined for each DNA sample using
GeneTools software (Syngene, Frederick, MD), and
converted into Shannon-Weiner (Shannon & Weaver
1963) richness (S), diversity (H′), and evenness
indices by soil type.

Statistical analysis
A weight of evidence statistical approach was
used to compare differences in the mean values of
all metrics determined from the four pooled soil of
each habitat type. The percent differences (PD), ttest P values, and the Cohen’s d effect size values
were used to suggest biologically meaningful differences between means, consistent with the recommendations for analysis of small sample sizes by
Di Stefano et al. (2005). We used approximate ttest P values ≤ 0.25, PD ≥ 10, and Hedge’s d values
≥ 0.7 (> 0.7 is considered a large effect size difference) to define biologically meaningful differences
in the means for this project.
Following analysis of the data for differences
between habitats, the data from all habitats was
aggregated (n = 8) and analyzed by Pearson’s Correlation methods to determine the strength of possible
relationships that will serve as soil ecosystem
condition indicator targets in future studies. Any
two factors resulting from this analysis with r
values > 0.443 or ≤ -0.443, with P values < 0.2
were considered critical correlations based on
standard tables of critical values of r. Since increases in the abundance of Basidiomycetes have been
identified as an indicator of soil complexity
(Anderson 2003; Baldrian 2006; Sinsabaugh 2010),
we calculated the correlation coefficients between
the RA of the laccase gene (indicating potential
lignin degradation by Basidomycetes) and the RA

HAFICH et al.

219

Table 3. A comparison of the physical, nutrient and microbial activity parameters in soil collected from
grasslands and adjacent secondary forests in Costa Rica.
Secondary Forest

Grassland

PD Sec. to Grass.

P value

Hedge’s d

Soil Physical Characteristics
pH

6.4 ± .2

6.3 ± 0.3

1.6

0.6

0.4

% Saturation

49.7 ± 3.9

52.3 ± 3.7

-2.6

0.51

0.53

Bulk Density (g cm-3)

0.7 ± 0.1

0.76 ± 0.05

-7.9

0.32

0.76

Soil Nutrient Characteristics
Mineral N (µg cm-3)

6.1 ± 0.9

4.4 ± 0.4

37.6

0.02

2.32

NO3-N (µg cm-3)

1.4 ± 0.3

0.5 ± 0.2

178.8

0.004

3.25

NH4-N (µg

cm-3)

4.6 ± 1.0

3.9 ± 0.3

18.7

0.23

0.95

24.3 ± 7.4

11.4 ± 4.4

113.2

0.02

2.12

Phosphate (µg cm-3)

8.8 ± 1.4

7.8 ± 0.5

13

0.22

0.98

Organic C (µg cm-3)

593 ± 35

532 ± 47

11.5

0.08

1.47

301 ± 34

219 ± 194

37.4

0.44

0.5

% N as Nitrate

Soil Carbon Use
Characteristics
Biomass C (µg cm-3)
Respiration (mg

g-1

h-1)

3.7 ± 0.4

2.8 ± 1.4

32.1

0.25

0.87

qCO2 (Resp/Cmic)

0.08 ± 0.01

0.14 ± 0.16

-42.9

0.48

0.53

Cmic/SOC

0.51 ± 0.05

0.43 ± 0.40

18.6

0.71

0.28

PD = percent difference in mean values; P = t-test P value; Hedge’s d effect size value.

Table 4. A comparison of the mean percent relative abundance (% RA) values (± standard deviation) of
Rhizobium 16S rRNA, nifH gene, laccase gene, and fungal 18S rRNA in secondary forest and Pentaclethradominant forest soils within the Maquenque National Wildlife Refuge in the Northern Zone of Costa Rica.
% RA
Rhizobium

% RA
nifH

% RA
Laccase

% RA
Fungus

% RA
Basidiomycetes

Grassland Soil (n = 4)

27.2 ± 9.4

25.1 ± 12.5

10.9 ± 0.04

6.5 ± 4.3

45.0 ± 12.7

Secondary Forest Soil (n = 4)

4.7 ± 1.6

12.6 ± 14.9

9.3 ± 5.4

24.9 ± 19.6

55.1 ± 9.2

PD

-82.7

-49.8

-7.9

283.1

21.9

P value

0.01

0.25

0.7

0.12

0.25

Cohen's d value

3.06

0.91

0.3

1.3

0.89

PD = percent difference in mean values; P = t-test P value; Hedge’s d effect size value.
of Basidiomycete rDNA with Cmic, Cmic/DOC, qCO2
as indicators of efficiency of C use (Anderson 2003);
and PO4, as some have suggested that the level of
microbial activity increases with increases in this
nutrient (Allison et al. 2007; Cleveland et al. 2004;
Cleveland & Townsend 2006; Cruz et al. 2009;
Eaton et al. 2011; Townsend et al. 2002).

Results
There were no meaningful differences between
the pH, percent water saturation, and bulk density

between the grassland and secondary forest sites.
The NO3-N, NH4-N, and total mineral N (TMN)
levels were greater in the secondary forest than
grassland soils, and the percent of TMN as NO3
was much greater in the secondary soils (Table 3) .
The levels of inorganic P and the respiration rate
were also greater in the secondary forest soils
(Table 3).
The abundance of the Rhizobium rDNA and
nifH gene DNA were greater in the grassland soils
than in the secondary forest soils, but the diversity, richness, and evenness of distribution of the

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LAND MANAGEMENT AND SOIL MICROBIAL COMMUNITIES

Table 5. RFLP-based Shannon-Weaver diversity indices for Rhizobium 16S rDNA, fungal 18S rDNA, and
Basidiomycete ITS DNA from grassland and secondary forest soils within the Maquenque National Wildlife
Refuge in the Northern Zone of Costa Rica. The Shannon-Weaver diversity index values are for richness (S),
diversity (H′), and evenness of distribution (E).
Rhizobium 16s rRNA
H′ index
S index
E index
Fungal 18s rRNA
H′ index
S index
E index
Basidiomycete ITS
H′ index
S index
E index

Grassland

Secondary Forest

PD

P value

d value

0.66 ± 0.36
2.75 ± 0.96
0.67 ± 0.27

1.55 ± 0.35
6.0 ± 2.58
0.89 ± 0.05

40.3
37.1
14.1

0.02
0.06
0.16

2.06
1.45
0.98

1.98 ± 0.27
9.33 ± 1.53
0.89 ± 0.06

1.72 ± 0.05
7.67 ± 1.15
0.85 ± 0.03

-7.0
-9.8
-2.3

0.11
0.13
0.28

1.16
1.07
0.73

0.7 ± 0.7
2.85 ± 1.71
0.52 ± 0.35

1.55 ± 0.35
5.50 ± 1.73
0.94 ± 0.01

37.8
92.9
28.8

0.06
0.07
0.06

1.34
1.54
1.41

PD = percent difference in mean values; P = t-test P value; Hedge’s d effect size value.

Table 6. Critical correlation results (defined as having r values > 0.443 or ≤ -0.443, with P values < 0.2) from
comparisons between certain parameters measured in the soil in secondary forest and grassland soils within
the Maquenque National Wildlife Refuge in the Northern Zone of Costa Rica. Pearson’s Correlations were made
between phosphate (PO4), microbial biomass (Cmic), carbon use efficiency metrics (Cmic/DOC and qCO2), and the
relative abundances (RA) of laccase gene and Basidiomycete ITS DNA. Only the r values and associated P
values (in parentheses) of the critical correlations are presented (NC = not critical).
PO4

Cmic

Cmic/DOC

RA Basidiomycetes

0.580(0.132)

0.715(0.046)

0.703(0.052)

-0.526(0.181)

RA Laccase

0.469(0.093)

0.453(0.196)

NC

-0.687(0.060)

Rhizobium rDNA were greater in the secondary
forest soils (Table 4). The abundance of both the
fungal and Basidiomycete rDNA were greater and
the diversity, richness, and evenness of the Basidiomycete rDNA was also greater in the secondary
forest soils (Table 4). However, the diversity, richness and evenness of the fungal rDNA was
somewhat greater in the grassland than secondary
soils, and there were no differences in the laccase
gene abundance and diversity between habitats
(Table 5).
Critical positive correlations were found between the RA of the Basidiomycete rDNA and P
and C biomass levels, and the ratio of C biomass to
DOC, and a critical negative correlation was found
between the Basidiomycete rDNA with the qCO2
(Table 6). Similar critical correlations were found
between the RA of the laccase gene DNA and these
same parameters, except there was no critical
correlation with the ratio of C biomass to DOC
(Table 6).

qCO2

Discussion
The evaluation of the soil ecosystem metrics
measured in this study have potential value for
assessment of ecosystem management, restoration,
and conservation strategies employed in the
secondary forests of the tropics, and for prediction
of changes in the amounts of C and N sequestered
into the biota (Buckley & Schmidt 2003; Ibekwe et
al. 2007; Nüsslein & Tiedje 1999; Oelbermann et
al. 2004; Wardle et al. 2004). The greater respiratory activity, fungal rDNA abundance, and Basidiomycete DNA abundance and diversity, phosphate, inorganic N, and organic C, suggest that the
secondary forest soil microbial community was
more complex, fungal dominant, and active than in
the grasslands, with more efficient use of C and
greater potential for C sequestration, which is
consistent with a more well-established and complex soil ecosystem (Anderson 2003). However, it is
also possible that there may be differences in these

HAFICH et al.

relationships that occur between wet and dry
season. To clarify this, we have begun a wet season
versus dry season study of the same plots and
parameters in the hope of determining the effect
that both land management and season has on
these soil ecosystems.
The greater amount of P in the secondary soils
being associated with greater microbial activity
and other indicators of a more complex soil ecosystem is consistent with the work of others in
similar forests (Allison et al. 2007; Anderson 2003;
Cleveland et al. 2004; Cleveland & Townsend 2006;
Cruz et al. 2009; Eaton et al. 2011; Townsend et al.
2002). After conversion of tropical rain-forest to
pasture, inorganic P declines, and organic P levels
either remain constant or increase (Townsend et
al. 2002), while microbial efficiency of organic C
use and P mineralization decreases in the long
term (Cleveland et al. 2002; Townsend et al. 2002).
Available P is a critical limitation of microbial C
use (Cleveland et al. 2002), and consequently
higher levels of P often result in increased
microbial biomass (Cleveland et al. 2004) and increased microbial respiration (Cleveland & Townsend 2006).
The greater amount of inorganic N and percent
of inorganic N as nitrate, along with the lower
abundance of the Rhizobium rDNA and nifH genes
in the secondary forest soils may be the result of
the recognized feedback inhibition effect that
elevated levels of inorganic N have on decreasing
the rates of N-fixation (Daimon & Yoshioka 2001;
King & Purcell 2005; Pons et al. 2007; Reed et al.
2007; Schulze 2004). These data also suggest the
possibility that there may be greater efficiency of
N-fixation, and perhaps rates of ammonium oxidation occurring in these secondary forest soils
(Booth et al. 2005; Sahrawat 2008; Webster et al.
2002). As well, other have shown lower rates of
nitrification occurred in pastures as compared to
forests in similar tropical areas (Carney et al.
2004; Cleveland et al. 2003; Reiners et al. 1994),
which can be due to a greater rate of assimilation
of N into the herbaceous biomass typically found in
grasslands, where N turnover is fastest (Booth et
al. 2005).
It is interesting that the greater levels of inorganic N were also associated with greater abundance of fungal and Basidiomycete rDNA. Although it has been well-established that higher
levels of inorganic N can inhibit soil fungi in
general, and the Basidiomycetes in particular
(Bittman et al. 2005; de Vries et al. 2007; Dighton
et al. 2004; Dijkstra et al. 2004; Hobbie 2008; Knorr

221

et al. 2005; Waldrop & Zak 2006), it is also recently
documented that nitrification can be carried out by
a number of species of fungi, especially in acidic
forest soils (Dighton et al. 2004; Hayatsu et al.
2008), and that shifts can occur in forest soils from
“nitrophobic” fungal species to more “nitrophilic”
species resulting in a fungal population less susceptible to inhibition by inorganic N (Dighton et al.
2004). Both of these fungal characteristics could
account for the greater fungal abundance levels in
the presence of soils with high levels of inorganic N.
Microbial community diversity and structure is
inextricably linked with nutrient parameters of
tropical soils and above-ground vegetation affected
by land use and management (Anderson 2003;
Cleveland et al. 2003; Reiners et al. 1994). Conversion of secondary forest to grassland soils has been
associated with decreases in the size and complexity of the microbial community (e.g. Bornemann & Triplett 1997; Cleveland et al. 2003). In
the current study, there was greater abundance
and dominance (i.e. lower E values) and lower
diversity of Rhizobium rDNA and nifH gene DNA
in the grasslands, and lower abundance of fungal
and Basidiomycete rDNA in the grassland soils
than the secondary forest soils. This, along with
the nutrient data, support the suggestion that the
secondary forests were more fungal dominant,
with more organic C being produced and used
more efficiently than in the grasslands.
An increased abundance of Basidiomycete fungi
and laccase activity associated with lignin degradation has been associated with and enhancement
of soil ecosystem complexity (Anderson 2003; Baldrian 2006; Sinsabaugh 2010). Our correlation analyses support this in that the biomass C and the
indicators of C use efficiency were correlated
appropriately with both the RA of Basidiomycete
rDNA and lacacse DNA. There was also a critical
correlation between the levels of phosphate and
the RA of Basidiomycete rDNA and laccase DNA,
which is consistent with earlier work conducted by
Eaton et al. (2011), who demonstrated an increase
in fungal activity in similar region tropical soils
when phosphate levels were increasing. These
results are encouraging for us and provide targets
for extended work on developing models to predict
soil quality in this region of Costa Rica.
Significant deforestation and land degradation
in the tropics has resulted in increased rates of
carbon dioxide and methane emissions to the atmosphere, loss of biodiversity, and a decrease in C
sequestration capacity. In response to this degradation, land management strategies in the tropics

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LAND MANAGEMENT AND SOIL MICROBIAL COMMUNITIES

are changing from intensive use to reforestation in
order to protect the large amount of C stored in
these soils (Jobbágy & Jackson 2000). The development of secondary forests is becoming an important strategy in the tropical terrestrial regions for
increasing the amount of soil carbon, carbon sequestration (Guo & Gifford 2002; Post & Kwon 2000;
Wright 2005), and forest ecosystem services in
general (see Chazdon 2008 for review). To confirm
the success of such strategies requires the assessment of the impact that these restoration methods
have on forest ecosystems. We suggest that a
combination of nutrient chemistry, microbial biomass
and activity measurements, and molecular microbiological evidence should be used to assess forest
restoration success in the tropics and also for development of predictive models to allow more informed land management decisions.

Acknowledgements
This work was funded by a grant from the
National Science Foundation (DBI-0452328) and
was conducted under the Costa Rican Government
Permit #063-2008-SINAC. The authors thank the
Summer 2009 NSF REU students for their assistance in sample collection and processing. We would
also like to thank Karen Siefer at Peninsula College
and Kurt Schmack and the staff members at the
Laguna del Lagarto Lodge in Boca Tapada, Alajuela,
Costa Rica for all their assistance in making this
project work.

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(Received on 20.05.2010 and accepted after revisions, on 15.07.2011)